The norepinephrine (NE) uptake mechanism is a process that has been widely studied and thoroughly characterized, and it is estimated that as much as 80% of the NE released during noradrenergic nerve stimulation is removed from the synapse by means of uptake. The importance of uptake is also reflected in the finding that certain psychotropic drugs, including tricyclic antidepressants, cocaine and amphetamine, alter brain NE uptake as their primary mode of action in altering mood and affect. In spite of the importance of uptake in synaptic function, very little is known about the nature of the carrier involved in transporting NE across the nerve terminal membrane. This proposal is the first attempt to isolate the NE uptake carrier by separating it from the other nerve membrane proteins for purposes of identification. A photoaffinity labeling procedure is to be used to attach radioactive NE covalently to the presumed carrier protein under ultraviolet light. This tight chemical bonding of the carrier to the radioactive form of its natural substrate (NE) will allow us to follow the labeled carrier throughout its solubilization and isolation procedures. The displacement of covalent binding by known inhibitors of NE uptake will be used to verify the labeling of the carrier. The covalently labeled carrier will be solubilized in SDS and subjected to slab gel electrophoresis to separate it from the other proteins in the nerve membranes. A fluorographic technique will allow us to distinguish the radioactive proteins(s) from the non-labeled membrane proteins as a means of identifying the pharmacologically defined NE uptake carrier. These experiments will elucidate the nature of the macromolecular species assumed to function as a carrier, and permit us to study its regulation in normal and in altered behavioral states, as found in animal models of affective disorders, drug abuse, hyperactivity and hypertension.